Oil of microorganisms rich in docosahexaenoic acid

ABSTRACT

The present invention relates to an oil of microorganisms rich in docosahexaenoic acid (DHA, C22:6n3), comprising more than 60% of DHA relative to the total mass of fat and to the use thereof for human or animal feed, in particular for feeding infants, children, or pregnant or lactating women.

FIELD OF THE INVENTION

The present invention relates to an oil of microorganisms rich indocosahexaenoic acid (DHA, C22:6n3), comprising more than 60% DHA inrelation to the total mass of fat and at least 80% triglycerides inrelation to the total mass of fat.

STATE OF THE ART

Oils containing DHA come from several sources, the most well known arefish, krill and microorganisms such as microalgae. Many strains ofmicroorganisms are known to produce PUFAs, in particular docosahexaenoicacid (DHA), arachidonic acid (ARA) or eicosapentaenoic acid (EPA), alsoidentified by the signs co 3 and o6. These PUFAs are widely used inindustry, in particular for human or animal food, or in cosmetics, andtheir industrial production has been continuously improved for manyyears (WO 1997/037032, WO 2001/054510, WO 2013/136028, WO 2015/004402,US 2017/016036, US 2017/335356). The criteria for selecting strainssuitable for industrial production are their high biomass productivity,a significant accumulation of triglycerides (TG) and a high PUFA contentin the fat. Today, many known industrial strains meet these first threecriteria, with a PUFA content in the fat of the order of 35%, or even50% in the best case.

However, there is a demand for concentrated oils with high PUFAcontents, namely for the supply of concentrated products such asconcentrated oil capsules that make it possible to reduce the number ofunit doses needed for an equivalent quantity of PUFA. To obtain oilswith a high PUFA content (for example above 55% DHA), the oils can beenriched by adding PUFA (US 2014/323569) and/or the oils areconcentrated by a process that converts triglycerides to ethyl estersinvolving the use of solvent such as ethanol. Ethyl esters are anartificial chemical form, they do not exist in nature. Thebioavailability of fatty acids in the form of ethyl esters is much lessthan in the form of triglycerides (Ghasemifard et al., 2014). Moreover,the process removes the vitamins and antioxidants present in the crudeoil. Consequently, the concentrated oil is more vulnerable to oxidation.

It is possible to convert these ethyl esters back to triglycerides,these are “reformed” triglycerides, in order to improve bioavailability.Antioxidants can also be added to increase the stability of the oil overtime. However, this concentrated oil is rather different from thenatural oil, it has undergone a number of transformation processes thathave changed its composition: fatty acid profile, vitamins, pigments andother antioxidant molecules, depriving the PUFAs of their naturalprotection. However, PUFAs are sensitive, in particulartemperature-sensitive, molecules which can convert cis bonds to transbonds (Tsuzuki W, 2012). It should also be noted that the re-formationof triglycerides is incomplete, the oil thus treated still contains avariable proportion of ethyl esters, which distinguishes it fromuntreated oil. The ethanol released during transesterification(conversion of ethyl esters to triglycerides) is generally removed byevaporation. Nevertheless, traces of ethanol remain in the concentratedoil.

Another reason for wanting to minimize oil treatment processes is theformation of contaminants such as monochloropropanediols (2-MCPDs,3-MCPDs) and glycidol and derivatives thereof (2-MCPD, 3-MCPD andglycidol fatty acid esters). The presence of these contaminants has beendetected in particular following the steps of purification anddeodorization of fish oil (Miyazaki and Koyama, 2017). There is atpresent little data available concerning the impact of concentrationprocesses on the formation of contaminants. However, the re-formation oftriglycerides from ethyl esters may lead to an increase in diglycerides,which are contaminant precursor compounds. The level of glycidol (andglycidol esters) is subject to regulation (EU) 2018/290/EC to limit itscontent in foods: the concentration must not exceed 1000 μg/kg in edibleoils except for edible oils intended for the preparation of foods forbabies and infants where the limit is 500 μg/kg. In preparations forbabies and infants, the maximum level is even lower: 75 μg/kg in powdersand 10 μg/kg in liquids. This level will be further reduced (50 and 6μg/kg, respectively) in 2019. The evaluation of maximum MCPDconcentrations is currently underway for oils and baby foods. For themoment, the regulation relates only to hydrolyzed vegetable proteins andsoy sauce (limit of 20 μg/kg).

It is therefore of interest to obtain an oil naturally rich in PUFAs,the composition of which is as close as possible to the liposolublesubstances of the producing microorganism, with a minimum ofcontaminants produced during treatments. This makes it particularlysuited to the integration of DHA in food products. Its very low 3-MCPDand glycidol content, combined with its high DHA content, makes it idealfor the preparation of foods intended for babies and infants.

Moreover, these concentrated oils are generally obtained by treatmentprocesses which are expensive and harmful to the environment.

Another known solution consists in generating genetically modifiedmicroorganisms to seek to promote the metabolic pathways of PUFAproduction (Hamilton & al., 2016) or mutants believed to produce moreDHA (WO 2017/09804). However, the choice of technical solutions islimited by the use made of the oils obtained, in particular in humanfood (Fedorova-Dahms I. & al., 2011).

There is a need for oils naturally concentrated in PUFA, which requireno treatments other than extraction methods, i.e., for which PUFAs areessentially in the form of triglycerides as produced by microorganisms.More particularly, there is a need for oils with a high PUFA content anda lower saturated fatty acid content. In addition to the question of oilquality, the interest in a low saturated fatty acid content is movingtoward a less viscous oil, easier to use at industrial levels, inparticular requiring less energy for its handling.

The invention meets this demand with an oil with a high DHA content,comprising at least 60% DHA in relation to the total mass of fat. Thisoil contains neither ethyl esters nor traces of solvent (ethanol ormethanol) and has a reduced content of 3-MCPD and glycidol (comparedwith oils containing more than 60% DHA currently on the market).

DISCLOSURE OF THE INVENTION

The present invention relates to a microbial oil which comprisesdocosahexaenoic acid (DHA), characterized in that it comprises at least80% triglycerides in relation to the total mass of fat, more than 60%DHA in relation to the total mass of fat and the saturated fatty acidcontent is less than 25% in relation to the total mass of fat.

It also relates to a diluted microbial oil which comprises a microbialoil rich in triglycerides and in DHA according to the invention, mixedwith another oil.

Another object of the invention is a biomass of microorganisms thatcomprises an oil rich in triglycerides and in DHA according to theinvention.

The invention also relates to the use of an optionally diluted oil richin triglyceride and in DHA according to the invention or of a biomassthat contains this oil for human or animal food, in particular for foodfor newborns, children, or pregnant or nursing women.

Another object of the invention is a food, characterized in that itcomprises an optionally diluted oil rich in triglycerides and in DHAaccording to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The oil according to the invention is a microbial oil which comprisesmore than 60% DHA in relation to the total mass of fat, advantageouslyat least 62% DHA, more advantageously at least 65% DHA, preferably morethan 67%, more preferentially at least 70%, even more preferentially 75%DHA in relation to the total mass of fat.

These characteristics of the oil according to the invention concern boththe oil as present in the biomass of microorganisms and the oilextracted from this biomass, whether crude or purified.

The invention also relates to a diluted oil, comprising the oilaccording to the invention mixed with another oil.

The invention also relates to a pharmaceutical, cosmetic or foodcomposition that comprises an oil according to the invention, whethercrude, refined or diluted.

The invention also relates to the use of an oil according to theinvention, crude, refined or diluted, or a biomass containing the oil,for human or animal food, in particular food for newborns, children, orpregnant or nursing women.

DETAILED DESCRIPTION OF THE INVENTION

The oil according to the invention is an oil of microbial origin,obtained from a biomass of microorganism cells grown under conditionsallowing both cell growth (to produce the biomass) and the production ofan oil with a high DHA content.

The oil according to the invention is a microbial oil which comprisesmore than 60% DHA in relation to the total mass of fat, advantageouslyat least 62% DHA, more advantageously at least 65% DHA, preferably morethan 67%, more preferentially at least 70%, even more preferentially atleast 75% DHA in relation to the total mass of fat.

Preferably the oil according to the invention has a high content ofunsaturated fatty acids in relation to saturated fatty acids. Theunsaturated fatty acids in the oil according to the invention areessentially DHA and DPA (docosapentaenoic acid, C22:5n6). The ARA(arachidonic acid, C20:4n6) content is generally less than 0.5%, or evenless than 0.3%, advantageously less than 0.1%. The EPA (eicosapentaenoicacid, C20:5n3) content is generally less than 1.5%, advantageously lessthan 1%, more advantageously less than 0.5%. The percentages of ARA andEPA are given in relation to the total mass of fat.

Advantageously, the combined DHA and DPA content is at least 70% inrelation to the total mass of fat, advantageously at least 75%, moreadvantageously at least 80%, and even more advantageously at least 85%in relation to the total mass of fat. In certain cases, total DHA+DPArepresents up to 90% of the total mass of fat. For the oils with thehighest DHA content, at least 70%, the combined DHA and DPA content isat least 80%, preferentially at least 85%.

For a DHA-rich oil according to the invention, the DHA/DPA ratio ispreferably at least 3, more preferentially at least 4, ranging from 4 to9. For the oils with the highest DHA content, at least 70%, the DHA/DPAratio is advantageously from 4 to 7.

The saturated fatty acid content is less than 25% in relation to thetotal mass of fat, or even less than 20%, more preferentially less than15%, even more preferentially less than 10%.

The saturated fatty acids are essentially palmitic acid (C16:0). Othersaturated fatty acids are present in a content less than 2%, or evenless than 1%, in particular pentadecylic acid (C15:0) or myristic acid(C14:0) or stearic acid (C18:0). Advantageously, C10 to C22 saturatedfatty acids other than palmitic acid are, independently of each other,present in trace amounts, each in a content of less than 0.1%, or evenabsent (0% taking into account the uncertainties of the methods ofanalysis), in particular for C10, C11, C12, C17, C20, C21 and C22saturated fatty acids. The percentages are given in relation to thetotal mass of fat.

The palmitic acid content is preferably less than 20% of the total massof fat, more preferentially less than 15%, even more preferentially lessthan 10%.

For the oils with the highest DHA content, at least 70%, the C10 to C22saturated fatty acid content is preferably less than 15%, morepreferentially less than 10%.

One way to measure the high DHA content of the oil according to theinvention and the low saturated fatty acid (SFA) content is to establisha DHA/SFA ratio.

It is advantageously at least 2.5, preferentially at least 3, morepreferentially at least 5, even more preferentially at least 6. It maygo up to at least 8 in some cases, or even at least 9. For the oils withthe highest DHA content, at least 70%, the DHA/SFA ratio is at least 4,preferentially at least 6, more preferentially at least 8, up to about9.

It is also possible to measure the high content of polyunsaturated fattyacids in relation to saturated fatty acids (SFA) by the (DHA+DPA)/SFAratio.

It is advantageously at least 2.5, preferentially at least 3, morepreferentially at least 4, even more preferentially at least 5. It maygo up to at least 8 in some cases, or even at least 9. For the oils withthe highest DHA content, at least 70%, the (DHA+DPA)/SFA ratio is atleast 5, preferentially at least 7, more preferentially at least 10, upto about 11 or more.

The oils according to the invention are essentially in the form oftriglycerides. Triglycerides represent at least 80% of the total mass offat, advantageously at least 90%, more advantageously at least 93% ofthe total mass of fat. The triglyceride content is for example analyzedby thin-layer chromatography (Jouet et al., 2003).

These characteristics of the oil according to the invention concern boththe oil as present in the biomass of microorganisms and the oilextracted from this biomass, whether crude or purified.

In certain cases, depending on the process used, the extraction of theoil from the biomass can lead to a slight increase in the DHA and DPAcontent, favoring the extraction of these PUFAs over saturated fattyacids of lower molecular weight. However, this concentration does notsubstantially modify the intrinsic properties of the oil contained inthe biomass, in particular the triglyceride content. In all cases, theoil according to the invention is an oil that has not undergonesubstantial modifications of its fatty acid content by the addition ofPUFA, for example in the form of esters, by concentration and/or by theremoval of saturated fatty acids such as palmitic acid.

The oil according to a particular embodiment of the invention containsmore than 10 mg of native carotenoids per kg of oil, or even more than30 mg/kg, preferentially more than 40 mg/kg, even more preferentiallymore than 60 mg/kg, or even at least 65 mg/kg. The carotenoids presentare predominantly astaxanthin and beta-carotenes. The oil contains morethan 20 mg/kg of astaxanthin, or even more than 30 mg/kg, morepreferentially more than 40 mg/kg. Canthaxanthin is also present but insmaller amounts. Other carotenoids such as lutein and zeaxanthin may bepresent but they are at the limit of detection of the method used. Theterm “native carotenoids” means that the carotenoids have not beenadded, they come from the same biomass as the oil and are extracted fromthis biomass at the same time as the oil. They are produced by thestrain under heterotrophic fermentation conditions, with no particularstimulus. These native carotenoids are therefore present throughout theprocess, protecting the fatty acids, in particular DHA, againstoxidation. The refining process can remove pigments, so the refined oilmay contain fewer carotenoids, if any.

The color of the oil is usually evaluated by measuring the Gardnerindex, according to the method described in standard AOCS Cc 13j-97(revised 2017) with a spectrophotometer. The measurement scale comprises18 grades, ranging from transparent (1) to dark red/brown (18). Somecarotenoids, including astaxanthin and beta-carotenes, show acoloration, more or less intense depending on their concentration. Theirpresence is thus reflected in a higher Gardner index. The oil accordingto a particular embodiment of the invention has a Gardner index higherthan 8 or even higher than 10, preferably between 12 and 17.

The Gardner scale is traditionally used to evaluate the aging of oilsbecause the oxidation of oils rich in polyunsaturated fatty acids(PUFAs) can result in a yellowing of the color (for a transparent oil),thus a higher Gardner value. However, the oxidation of PUFA-rich oils ismore precisely measured by the anisidine index and the peroxidationindex. The oils according to the invention have both low anisidine andperoxide indexes, which guarantee a low oxidation product, and a highGardner index, due to the presence of carotenoids. The oils according tothe invention have an anisidine index of less than 5, or even less than2, preferably less than 1.5, and a peroxidation index of less than 5, oreven less than or equal to 1, preferably less than or equal to 0.5.

The oils according to the invention have a fairly low meltingtemperature, which decreases in correlation with the increase in DHAcontent. The melting temperature is measured according to standard ISO6321. Indeed, the oils, with more than 600 mg of DHA/g of fatty acids(or about more than 62% DHA) have a melting temperature below 20° C.,even less than or equal to 5° C. They are therefore liquid at roomtemperature. The oils with more than 700 mg of DHA per g of fatty acids(or about more than 73% DHA) have a melting temperature below −5° C. Alow melting temperature facilitates storage and handling (pumping inparticular), since it is possible to store the oil in liquid form whilerefrigerating it in order to limit aging. Oils that freeze duringstorage must be warmed up for sampling and for incorporation intomixtures. However, temperature is a factor that accelerates oxidation.

This property is also reflected in the viscosity value, measured by aviscometer at 22° C. (Viscoman, Gilson). The oils according to theinvention have a viscosity value at room temperature of less than orequal to 50 Pa·s, or even of less than 40, preferably of less than 30.

The oils according to the invention are obtained by culturingmicroorganisms that produce DHA-rich oils. The strains of microorganismswhich make it possible to obtain such oils are industrial strains, i.e.,according to the invention, strains the fat content of which representsat least 45% of the dry matter, preferentially at least about 50% of thedry matter, and which have a growth capacity at a cell density of atleast 50 g/L, preferentially at least 70 g/L, more preferentially atleast 100 g/L.

The person skilled in the art is familiar with industrial strains ofPUFA-producing microorganisms mainly among thraustochytrids,dinoflagellates, diatoms, eustigmatophytes, in particular microorganismsof the genera Crypthecodinium, Schizochytrium, Thraustochytrium orAurantiochytrium for the production of DHA.

The analysis of PUFA content in the fat is carried out according to thestandard methods of the skilled person, in particular described in thefollowing article: Gas Chromatographic Quantification of Fatty AcidMethyl Esters: Flame Ionization Detection vs. Electron Impact MassSpectrometry, Dodds et al., Lipids, Vol. 40, no. 4 (2005).

More particularly, mention may be made of the strains Aurantiochytriummangrovei CCAP4062/7 and CCAP4062/8 and Schizochytrium sp. CCAP4087/7which produce oils comprising more than 60% DHA in relation to the totalmass of fat. The invention also relates to those strains capable ofproducing oils comprising more than 60% DHA.

The processes for the industrial culture of microorganisms for theproduction of a fermenting must which will then be used to produce oilare well known to the person skilled in the art, whether in autotrophic,heterotrophic or mixotrophic mode. Industrial culture in heterotrophicor mixotrophic mode allows cell densities of at least 50 g/L,preferentially at least 70 g/L, more preferentially at least 100 g/L.

According to the invention, “industrial culture” means a culture of thestrains in a culture medium suitable for their growth and for PUFAproduction and in a volume suitable for the production of sufficientamounts to address a market.

These industrial cultures are carried out by fermentation in adiscontinuous “batch” mode, a semi-continuous “fed batch” mode or acontinuous mode. The fermenters have volumes which can range from 1000 Lto more than 200 m³.

The suitable culture medium is preferably a chemically defined culturemedium that comprises a carbon source, a nitrogen source, a phosphorussource and salts. “Chemically defined culture medium” means a culturemedium in which the content of each element is known. Advantageously,the medium does not include rich or complex organic matter. Rich orcomplex organic matter means unpurified organic matter in the form ofmixtures for which the exact composition and concentrations of thevarious components of the mixture are not accurately known, notcontrolled, and may show significant variability from batch to batch. Asexamples of rich or complex organic matter, mention may be made of yeastextracts or peptones which are products of a protein hydrolysisreaction, or rich mineral matter such as marine mineral salts or othercomplex growth agents, not having a fixed concentration of each of theircomponents.

Generally, industrial culture processes comprise a growth step topromote biomass production, followed by an accumulation step to promotethe production of fat and PUFA in particular. This is notably the casefor the process described in patent application WO 2001/054510. Morerecently, processes have been described using culture conditions thatconcomitantly promote the production of biomass and that of PUFA.Particular mention may be made of the culture methods described inapplications WO 2012/035262, WO 2015/004402 and WO 2015/004403. Ofcourse, the skilled person will be able to adapt the culture conditions,in particular the composition of the medium, the conditions for addingnutrients during the culture, the temperature, oxygenation cycles andthe lighting conditions to promote biomass production.

The temperatures of industrial culture are advantageously greater than17° C.

According to the invention, “biomass” advantageously means a set ofmicroorganism cells produced by their culture, in particular by themethods described above, cells which may or may not have retained theirphysical integrity. It is therefore understood that said biomass mayinclude a quantity of degraded microorganism cells from 0% to 100%.“Degraded” means that the physical integrity of said microorganism cellsmay have been altered, such as lysed microorganisms, resulting forexample from a process of homogenization or enzymatic lysis. Onceproduced, this biomass can be raw, just separated from its culturemedium, dried or not, degraded or not.

The biomass, depending on whether it is dried or not, totally orpartially, can have a moisture content of 1% to 90%.

The invention therefore also relates to a biomass of microorganismscomprising an oil as previously defined.

According to a first embodiment, the biomass has a moisture content of70% to 90%, preferentially 80% to 85%. This is particularly the casewhen it essentially consists of optimized industrial microorganismscultivated after filtration of the fermenting must to separate thecultivated microorganisms from the culture medium, before drying.

According to another embodiment of the invention, the biomass is dried,totally or partially, and has a moisture content of 1% to 10%,preferentially 2% to 7%.

The biomass may be packaged for storage or for use as such, for exampleas a food supplement or food for human or animal consumption.

The methods for isolating an oil according to the invention from abiomass produced by the culture of microorganisms are well known to theperson skilled in the art. Particular mention may be made ofsolid-liquid extraction which is based on the use of a solvent (liquidphase) to extract the oil contained in the dried biomass (solid phase)by sprinkling or maceration; liquid-liquid extraction which is based onthe separation of the aqueous phase from the oil after preliminary lysisof the cells and then decanting or centrifugation. Preferably theextraction is done without organic solvents. Particular mention may bemade of the applications WO 01/53512, WO 02/10423, WO 2014/122092, WO2015/092546 and WO 2015/095694.

Mention may also be made of a preferred method for improving the fatextraction yields from microorganisms for PUFA-rich oils. This methodconsists in carrying out cell lysis at a first temperature, the latterbeing continued at a second temperature lower than the first, thenmechanical separation of the oil from the lysed biomass (filtration,decanting).

Cell lysis is done by enzymatic or mechanical lysis (grinding). Thetemperature of the first part of lysis is preferably at least 50° C.while remaining below temperatures that would degrade the composition ofthe oils in addition to promoting cell lysis, i.e., temperatures below80° C., preferably at most 70° C.

The enzymes that may be used are known, in particular described inWO2015/095688, WO2011/153246, U.S. Pat. No. 6,750,048 and WO2015/095694,in particular proteases or cellulases such as the enzymes marketed bythe firm Novozyme under the names Alcalase 2.5 L, Alcalase 2.4 L,Novozym 37071, Flavourzyme 1000 L, Novozym FM 2.4 L, Protamex,Viscozyme. The conditions of use are those recommended by the supplier,the temperature being that recommended for optimal enzyme activity, atleast 50° C. and up to 70° C., preferably about 65° C. Advantageously,enzymatic lysis is carried out in an oxygen-poor atmosphere.

Mechanical lysis methods are also well known, in particular by ballmill, mixer-disperser, high-pressure homogenizer pin mill or impactmill, ultrasonic, pulsed electric fields. Particular mention may be madefor the ball mill: Netzsch/Discus-1000; WAB/ECM-AP60; for thehigh-pressure homogenizer: GEA/Ariete; for the mixer-disperser:Silverson/700-X, for the pin mill: Hosakawa/Contraplex; for the impactmill: Netzsch/Condux.

The first part of the lysis is carried out under the usual conditionsrecommended by the state of the art for cell lysis, in particular interms of the duration of the enzymatic lysis or the grinding cycles.

The lysis continuation step completes the lysis by modifying theimplementation conditions without having to extract the lysed biomassbeforehand. The lysis temperature in this second part is at least 10° C.lower than that of the first part. Preferably, the temperature of thesecond part of lysis is less than or equal to 40° C., advantageouslyranging from 5° C. to 40° C. This second part of lysis at a lowertemperature, or end of lysis, is advantageously carried out for at least30′, advantageously up to 30 h.

The mechanical separation of an oil from a lysed biomass is also wellknown to the skilled person, as a gravity separation, in particular bycentrifugation as described in patent application WO 01/53512. It isalso possible to use continuous separation, in particular by centrifugalplate separator. Such separators are known to continuously extract oilsfrom complex media comprising solid residues and water, as described inpatent application WO 2010/096002, in particular marketed by thecompanies Alfa Laval, Flottweg or SPX Flow Technology Santorso, amongothers. This continuous separation step is preferred in the process usedto obtain the oil according to the invention.

The oil obtained is generally an oil called crude oil, which can be usedas is or can be refined, in particular to facilitate its storage, bypreventing it from becoming rancid, or to change its color so as to makeit more acceptable to a consumer. These refining steps are well known tothe person skilled in the art, in particular degumming, clarificationand deodorization. They remove (totally or partially) phospholipids,pigments, volatiles and free fatty acids. In fact, these methods do notsubstantially modify the relative content of fatty acids, saturated orunsaturated, nor the triglyceride content of the refined oil obtainedcompared with the crude oil.

The invention also relates to a packaged oil comprising a container ofsuitable volume to contain said oil, the oil being a DHA-rich oil aspreviously defined, crude or refined, and packaged in a quantity of oilgreater than 1 L, advantageously in a quantity of oil greater than 10 L,more particularly in a quantity of oil of the order of 220 L, and moreparticularly in a quantity of oil of the order of 20 m³.

Any container capable of holding the volume of oil or biomass andprotecting them for their storage and transportation may be used by theperson skilled in the art. Advantageously, the volume of the containerwill be equal to or substantially greater than that of the oil orbiomass packaged in such a way as to limit the presence of air in thecontainer and limit oxidation. The container will be advantageouslyopaque in order to avoid the degradation of the product by light rays,in particular UV rays. Advantageously, the container will be airtight sothat any volume not occupied with oil or biomass can be filled with aninert gas.

The oil according to the invention can be mixed with other oils fortheir final use. This dilution changes the overall content of DHA andother unsaturated fatty acids in the composition of the diluted oil. Itremains possible, however, to identify in the final oil, in view of thefatty acid profile of the oil used for dilution, the relativepercentages of fatty acids that come from the DHA-rich oil according tothe invention and from the dilution oil.

The invention therefore also relates to a diluted oil, comprising theoil according to the invention mixed with another oil. The oils used todilute the DHA-rich oil according to the invention are generally andpreferably vegetable oils suitable for human or animal food consumption.Particular mention may be made of sunflower, rapeseed, soybean, walnut,sesame, hemp, hazelnut, argan, olive, linseed or any other oil suitablefor food use. The added oil may also be an oil containing other PUFAs,in particular ARA and/or EPA, in particular other oils of microbialorigin or fish oils.

The invention also relates to a composition that comprises an oilaccording to the invention, whether crude, refined or diluted, or thatcomprises the biomass according to the invention.

A composition according to the invention may comprise one or moreexcipients. An excipient is a component, or mixture of components, whichis used in the present invention to give desirable characteristics tothe composition for its storage and use, including foods andpharmaceutical, cosmetic and industrial compositions. An excipient maybe described as a “pharmaceutically acceptable” excipient when it isadded to a pharmaceutical composition whose properties are known fromthe pharmacopoeia to be used in contact with human and animal tissueswithout excessive toxicity, irritation, allergic reaction or othercomplications. Different excipients may be used such as an organic ormineral base, an organic or mineral acid, a pH buffer, a stabilizer, anantioxidant, an adhesion agent, a release agent, a coating agent, anouter phase component, a controlled release component, a surfactant, ahumectant, a filler, an emollient, or combinations thereof.

Depending on their destination, the compositions according to theinvention are in particular pharmaceutical, cosmetic, nutraceuticalcompositions or foods.

Foods are intended for both humans and animals and include solid, pastyor liquid compositions. Particular mention may be made of common foods,liquid products, including milks, beverages, therapeutic beverages andnutritional beverages, functional foods, supplements, nutraceuticals,preparations for infants, including preparations for premature infants,foods for pregnant or nursing women, foods for adults, geriatric foodsand animal feeds.

The DHA-rich oil according to the invention, whether crude or refined,or the biomass containing it can be used directly as or added as anadditive in an oil, a spread, another fat ingredient, a beverage, asoy-based sauce, dairy products (milk, yogurt, cheese, ice cream),bakery products, nutritional products, for example in the form ofnutritional supplement (in capsule or tablet form), vitamin supplements,food supplements, powders to be diluted for beverages, such as energydrinks or milk powders for infant formulations, finished orsemi-finished powdered food products, etc., according to the known usesof the person skilled in the art.

Animal feeds are also known to the person skilled in the art. They arein particular intended for farm animals, such as cows, pigs, chickens,sheep, goats or in fish farming for shellfish or farmed fish.

Pharmaceutical compositions comprising a DHA-rich oil are also known tothe skilled person, the oil being used alone or in combination withother medicinal products.

The oil according to the invention, crude or refined, or the biomasscontaining it, may be formulated in the form of single-dosecompositions, in particular in the form of tablets, capsules, powders,granules, suitable for oral administration.

The advantage of the DHA-rich oil according to the invention, whethercrude or refined, or of the biomass containing it, is that it can beused in smaller amounts in these mixtures and compositions.

The invention also relates to the use of an oil according to theinvention, crude, refined or diluted, or the biomass containing it, forhuman or animal food, in particular food for newborns, children, orpregnant or nursing women.

Such uses are well known to the person skilled in the art, in particulardescribed in patent application WO 2010/107415 and on the website of thefirm DSM(https://www.dsm.com/markets/foodandbeverages/en_US/products/nutritional-lipids/life-dha.html).

EXAMPLES Example 1: Fatty Acid Profile of High DHA Content Biomass ofThraustochytrids

Strains of thraustochytrids (Aurantiochytrium mangrovei—FCCB1897,FCCB1800, CCAP4062/8) are grown in Erlenmeyer flasks in ATCC 790(modified) culture medium. Similar results are obtained with strains ofSchizochytrium sp. (in particular with strain CCAP4087/7).

Once the cultures are in the stationary phase, the biomass is recoveredby centrifugation and then freeze-dried before analysis of the fattyacid composition of the biomass by GC-FID (method adapted from standardISO 12966-2).

Composition of the Modified ATCC 790 Medium:

Yeast extract 5.0 g/L Peptone 5.0 g/L D+-Glucose 30.0 g/L  Sea salts  20g/L

Table 1 represents the composition of fatty acids contained in thebiomass. The results are expressed as a percentage of the total fattyacid content. SFAs are saturated fatty acids.

TABLE 1 FCCB1897 FCCB1800 CCAP4062/8 C10:0 0.0 0.0 0.0 C11:0 0.0 0.0 0.0C12:0 0.0 0.0 0.0 C13:0 0.0 0.0 0.0 C14:0 0.2 0.3 0.3 C14:1 0.0 0.0 0.0C15:0 0.6 1.0 1.7 C15:1 0.0 0.0 0.0 C16:0 6.8 6.5 8.3 C16:1 0.0 0.0 0.0C16:2 0.0 0.0 0.0 C16:3 0.0 0.0 0.0 C16:4 0.0 0.0 0.0 C17:0 1.1 1.4 1.5C17:1 0.3 0.3 0.2 C18:0 0.5 0.5 0.4 C18:1 0.0 0.0 0.0 C18:2 0.0 0.0 0.0C18:3n3 0.1 0.1 0.1 C18:3n6 0.1 0.1 0.0 C18:4n3 0.3 0.5 0 .4 C20:0 0.10.1 0.0 C20:4n6 (ARA) 0.3 0.3 0.3 C20:5n3 (EPA) 1.1 1.0 1.2 C21:0 0.00.0 0.0 C22:0 0.0 0.0 0.0 C22:5n3 (DPAn3) 0.4 0.3 0.4 C22:5n6 (DPAn6)16.7 16.5 16.8 C22:6n3 (DHA) 70.9 70.9 68 DHA + DPA 88.0 87.8 85.2 SFA7.7 7.8 10.2 DHA/DPA 4.2 4.2 4.0 DHA/SFA 9.2 9.1 6.7 (DHA + DPA)/SFA11.4 11.3 8.4

Example 2: Fermenter Cultures of High DHA Content Strains

The cultures are carried out in fermenters (bioreactors) from 1 to 5 Luseful with dedicated automated systems and supervision by computerstation. They are carried out using two strains of Aurantiochytriummangrovei and with two different culture protocols. The system isregulated at pH 5 via the addition of base (NH₄OH for example b1 and b2and with NaOH for example a) with pH adjustment carried out throughoutthe culture period, and providing a nitrogen supply (in the context ofexamples b1 and b2). The culture temperature was set at 30° C. then 22°C. and finally 18° C. at the end of the culture.

Strain CCAP4062/7 is used for example a and b1 whereas strain FCCB1897is used for example b2.

The composition of the culture media is given in Table 2.

TABLE 2 a b1 and b2 CaCl2, 2H2O 0.55 0.55 g/L MgSO4, 7H2O 4-8 4-8 g/LH3B03 0.00875-0.175  0.00875-0.0175  g/L K2SO4 2.08 0.00 g/L KH2PO4 4.004.00 g/L Na4EDTA, 2H2O 0.12 0.12 g/L FeSO4, 7H2O 0.04 0.04 g/L (NH4)2SO49.00 1.00-2.00 g/L MnCl2, 4H2O 0.0108 0.0108 g/L ZnSO4, 7H2O 0.01080.0108 g/L CoCl2, 6H2O 0.000108 0.000108 g/L Na2MoO4, 2H2O 0.0001080.000108 g/L Na2SeO3 1.73E−07 1.73E−07 g/L CuSO4, 5H2O 0.0072 0.0072 g/LNiSO4, 6H2O    0-0.0056    0-0.0056 g/L Thiamine 0.0320 0.0320 g/LVitamin B12 0.0005 0.0005 g/L Pantothenate 0.0108 0.0108 g/L DefoamerBiospumex 153K 0.40 0.40 mL/L Glucose, 1 H2O 55.00 55.00 g/L

Additions of glucose in the form of an enrichment solution are made witha carbon:nitrogen:phosphorus (CNP) molar ratio of 533:11:1 (example a)or with a solution composed only of glucose (examples b1 and b2).

Culture Monitoring:

The total biomass concentration is monitored by measuring the dry mass(filtration on Whatman GF/F filter then oven drying, at 105° C., for aminimum of 24 h before weighing). Fatty acid analyses are carried outaccording to a method adapted from ISO 12966-2 for the biomass, andaccording to the European Pharmacopoeia 9.0 (2.4.29.) for the oils.

The fatty acid profiles of the biomasses obtained with conditions a, b1and b2 are given in Table 3. The results are expressed as a percentageof the total fatty acid content.

TABLE 3 a b1 b2 C10:0 0.0 0.0 0.0 C11:0 0.0 0.0 0.0 C12:0 0.0 0.0 0.0C13:0 0.0 0.0 0.0 C14:0 0.8 1.2 0.3 C14:1 0.0 0.0 0.0 C15:0 0.0 0.1 1.7C15:1 0.0 0.0 0.0 C16:0 13.6 19.6 10.9 C16:1 0.1 0.2 0.2 C16:2 0.0 0.00.0 C16:3 0.0 0.0 0.0 C16:4 0.0 0.0 0.0 C17:0 0.0 0.0 0.0 C17:1 0.0 0.00.3 C18:0 0.5 0.6 0.6 C18:1 0.2 0.3 0.4 C18:2 0.0 0.0 0.0 C18:3n3 0.20.1 0.2 C18:3n6 0.1 0.1 0.1 C18:4n3 0.3 0.3 0.3 C20:0 0.1 0.1 0.1C20:4n6 (ARA) 0.1 0.0 0.1 C20:5n3 (EPA) 0.4 0.6 0.6 C21:0 0.0 0.1 0.0C22:0 0.1 0.0 0.0 C22:5n3 (DPAn3) 0.2 0.0 0.2 C22:5n6 (DPAn6) 12.9 9.612.7 C22:6n3 (DHA) 66.6 62.5 70.1 DHA + DPA 79.7 72 83 SFA 15.1 22 11.4DHA/DPA 5.2 6.5 5.5 DHA/SFA 4.4 2.9 6.2 (DHA + DPA)/SFA 5.3 3.3 7.3

Example 3: Culture Under Industrial Conditions

High DHA content strains produce a biomass with a similar fatty acidcomposition when grown in industrial size fermenters, such as 10 m³ or180 m³ tanks, under conditions similar to example 2, with culture mediumb and glucose additions in the form of an enrichment solution are madewith a carbon:nitrogen:phosphorus (CNP) molar ratio of 533:0.4:1.

The fatty acid profiles of the CCAP4062/7 strain biomass after culturein 10 m³ and 180 m³ tanks are given in Table 4. The results areexpressed as a percentage of the total fatty acid content.

TABLE 4 10 m³ 180 m³ C10:0 0.0 0.0 C11:0 0.0 0.0 C12:0 0.0 0.0 C13:0 0.00.0 C14:0 0.7 0.5 C14:1 0.0 0.0 C15:0 0.0 0.0 C15:1 0.0 0.0 C16:0 18.113.6 C16:1 0.0 0.0 C16:2 0.0 0.0 C16:3 0.0 0.0 C16:4 0.0 0.0 C17:0 0.00.0 C17:1 0.0 0.0 C18:0 0.8 0.0 C18:1 0.0 0.0 C18:2 0.0 0.0 C18:3n3 0.30.3 C18:3n6 0.0 0.0 C18:4n3 0.0 0.0 C20:0 0.0 0.0 C20:4n6 (ARA) 0.2 0.1C20:5n3 (EPA) 0.9 0.4 C21:0 0.0 0.0 C22:0 0.0 0.0 C22:5n3 (DPAn3) 0.00.0 C22:5n6 (DPAn6) 11.2 13.4 C22:6n3 (DHA) 63.9 66.8 DHA + DPA 75.180.2 SFA 19.6 14.7 DHA/DPA 5.3 4.8 DHA/SFA 3.3 4.5 (DHA + DPA)/SFA 3.85.5

Example 4: Extraction of Oil from the Biomass of High DHA ContentStrains

The oil is extracted from the biomass of example 3 (180 m³ tank)according to a method described in WO2015/095694 (example 9). The fattyacid composition of the oil is similar to that of the biomass, given inTable 4.

Example 5: Extraction of Oil from the Biomass of High DHA ContentStrains

The extraction of the biomass produced under the same conditions as inexample 3 is carried out by following the sequence

-   (a) cell lysis by enzymatic means (e.g., with Alcalase 2.5 L or    Alcalase 2.4 L or Novozym 37071 from Novozymes) for 4 h at a    temperature of 65° C.,-   (b) continuation of the lysis by lowering the temperature between 5    and 40° C., for a duration comprised between 30 minutes and 30 h,-   (c) mechanical oil separation by centrifugal plate separator.

The extraction yield is 60% lipids extracted from the biomass.

The lipid profile of the oil extracted from the biomass is given inTable 6. The results are expressed as a percentage of the total fattyacid content.

TABLE 6 10 m³ 180 m³ C10:0 0.0 0.0 C11:0 0.0 0.0 C12:0 0.0 0.0 C13:0 0.00.0 C14:0 0.3 0.0 C14:1 0.0 0.0 C15:0 0.0 0.0 C15:1 0.0 0.0 C16:0 15.38.3 C16:1 0.0 0.0 C16:2 0.0 0.0 C16:3 0.0 0.0 C16:4 0.0 0.0 C17:0 0.00.0 C17:1 0.0 0.0 C18:0 0.4 0.4 C18:1 0.0 0.0 C18:2 0.0 0.0 C18:3n3 0.00.0 C18:3n6 0.0 0.0 C18:4n3 0.0 0.0 C20:0 0.0 0.0 C20:4n6 (ARA) 0.0 0.0C20:5n3 (EPA) 0.5 0.2 C21:0 0.0 0.0 C22:0 0.0 0.0 C22:5n3 (DPAn3) 0.00.0 C22:5n6 (DPAn6) 11.6 15.3 C22:6n3 (DHA) 71.1 75 DHA + DPA 82.7 90.3SFA 16 8.7 DHA/DPA 5.9 4.8 DHA/SFA 4.4 8.6 (DHA + DPA)/SFA 5.2 10.4

Example 6: Oil Quality: Antioxidants and Contaminants

Several heterotrophic fermentations are carried out according to theconditions of example 3. The oil is extracted from the fermenting mustaccording to the conditions of example 5. Carotenoids are measured inthe extracted oil, by LC/DAD, according to the following methods:Astaxanthin (including ester forms), Reference Method: DSM Ver. 1.52009; Beta-carotene (sum of cis- & trans-), saponified, Referencemethod: EN 12823-2:2000; Canthaxanthin, Reference method: Roche IndexNo. 2264; Lutein & Zeaxanthin, Reference Method: Roche Index No. 2264.

TABLE 7 Batch No. A B C D E DHA (mg/g FA) 670 674 663 700 707 DHA (% MG)70.6 71.1 70.6 72.2 72.4 Beta-carotenes (mg/kg 14 14.1 13.4 11.7 11.9FA) Astaxanthin (mg/kg FA) 46.1 41.4 38.2 28 25.9 Astaxanthin esters 24.5 4.7 4.8 5 (mg/kg FA) Canthaxanthin (mg/kg 3.5 3.3 3.2 3.7 3.8 FA)Total carotenoids 65.6 63.3 59.6 48.3 46.6 (mg/kg FA) Gardner index 14.816.7 >18 12.3 12.3 Anisidine index 1.17 1.13 0.54 1.35 1.97 Peroxideindex (meq/kg) 1 0.1 0.1 1 0.1

Contaminants such as glycidol and 2- and 3-MCPDs are also assayed in thesame batches.

TABLE 8 Batch No. A B C D E DHA (mg/g FA) 670 674 663 700 707 DHA (% MG)70.6 71.1 70.6 72.2 72.4 Glycidyl esters (glycidol <100 45 <100 <100<100 μg/kg) 2-MCPD (free and esters, <100 <100 <100 <100 <100 μg/kg)3-MCPD (free and esters <100 210 <100 <100 <100 and glycidyl esters)μg/kg

Example 7: Viscosity

The viscosity of the oil produced and extracted under conditions similarto example 5 is measured by a viscometer (Viscoman, Gilson) at differenttemperatures. The melting temperature is evaluated according to standardISO 6321.

TABLE 9 DHA (mg/g Viscosity Viscosity Viscosity Melting fatty acids - at22° C. at 8° C. at −20° C. temperature % MG) (Pa · s) (Pa · s) (Pa · s)(° C.) Sample 1 582-62 50 >2000 >2000 19 Sample 2 634-67 39 >2000 >200011 Sample 3 689-73 31 41 >2000 <−5° C.

REFERENCES

-   EP 0 223 960; EP 1 001 034-   US 2014/323569, US 2017/016036, US 2017/335356-   WO 1994/008467; WO 1997/037032; WO 2001/054510; WO 03/049832; WO    2010/107415; WO 2012/035262; WO 2013/136025; WO 2013/136028; WO    2014/146098; WO 2015/004402; WO 2015/004403; WO 2015/150716; WO    2016/030631, WO 2017/094804-   Fedorova-Dahms I. & al., Safety evaluation of DHA-rich algal oil    from Schizochytrium sp, Food and Chemical Toxicology, 2011, 49,    3310-3318-   Folch J, et al., A simple method for the isolation and purification    of total lipides from animal tissues. J Biol Chem. 1957 May;    226(1):497-509-   Hamilton M. & al., Heterotrophic Production of Omega-3 Long-Chain    Polyunsaturated Fatty Acids by Trophically Converted Marine Diatom    Phaeodactylum tricornum, Marine Drugs, 2016, 14, 53-   Omega-3 long chain fatty acid “bioavailability”: a review of    evidence and methodological considerations.-   Ghasemifard S, Turchini G M, Sinclair A J. Prog Lipid Res. 2014    October; 56:92-108. doi: 10.1016/j.plipres.2014.09.001. Epub 2014    Sep. 16. Review.-   Wakako TSUZUKI, Study of the Formation of trans Fatty Acids in Model    Oils (triacylglycerols) and Edible Oils during the Heating Process,    JARQ 46 (3), 215-220 (2012)-   Kinuko Miyazaki* and Kazuo Koyama, An Improved Enzymatic Indirect    Method for Simultaneous Determinations of 3-MCPD Esters and Glycidyl    Esters in Fish Oils, J. Oleo Sci. 66, (10) 1085-1093 (2017)-   Jouhet J., Marechal E., Bligny R., Joyard J., Block M. A. (2003).    Transient increase of phosphatidylcholine in plant cells in response    to phosphate deprivation. FEBS Lett. 544 63-68.

1. A microbial oil extracted from a microbial biomass which comprisesdocosahexaenoic acid (DHA), wherein said microbial oil comprises atleast 80% triglycerides in relation to the total mass of fat, more than60% DHA in relation to the total mass of fat, and the saturated fattyacid content is less than 25% in relation to the total mass of fat, andwherein the said microbial oil has not undergone substantialmodification of its fatty acid content by the addition ofpolyunsaturated fatty acids, by concentration and/or by the removal ofsaturated fatty acids.
 2. The microbial oil according to claim 1,wherein said microbial oil comprises at least 65% DHA in relation to thetotal mass of fat.
 3. The microbial oil according to claim 1, whereinsaid microbial oil comprises docosapentaenoic acid (DPA) and thecombined DHA and DPA content in said microbial oil is at least 70% inrelation to the total mass of fat.
 4. The microbial oil according toclaim 1, wherein said microbial oil comprises at least 70% DHA.
 5. Themicrobial oil according to claim 4, wherein said microbial oil comprisesdocosapentaenoic acid (DPA) and the combined DHA and DPA content in saidmicrobial oil is at least 80% in relation to the total mass of fat. 6.The microbial oil according to claim 1, wherein said microbial oilcomprises docosapentaenoic acid (DPA) and the DHA/DPA ratio in saidmicrobial oil is at least
 4. 7. The microbial oil according to claim 1,wherein the saturated fatty acid content in said microbial oil is lessthan 15% in relation to the total mass of fat.
 8. The microbial oilaccording to claim 1, wherein said microbial oil has a viscosity at roomtemperature of 50 Pa·s or less.
 9. The microbial oil according to claim8, wherein said microbial oil has a viscosity of less than 30 Pa·s. 10.A diluted microbial oil, wherein said diluted oil comprises a microbialoil according to claim 1, mixed with another oil.
 11. A biomass ofmicroorganisms, obtained by industrial culture of the saidmicroorganisms and comprises an oil comprising docosahexaenoic acid(DHA), wherein in said oil at least 80% triglycerides in relation to thetotal mass of fat, more than 60% DHA in relation to the total mass offat, and the saturated fatty acid content is less than 25% in relationto the total mass of fat.
 12. (canceled)
 13. A food, wherein said foodcomprises an oil according to any one of claims 1 to
 10. 14. Themicrobial oil according to claim 1, wherein said microbial oil comprisesless than 0.5% of ARA (arachidonic acid) in relation to the total massof fat.
 15. The microbial oil according to claim 1, wherein saidmicrobial oil comprises less than 1.5% of EPA (eicosapentaenoic acid) inrelation to the total mass of fat.
 16. The microbial oil according toclaim 1, wherein said microbial oil has a melting temperature below −5°C.
 17. The biomass of microorganisms according to claim 11, wherein themicrobial oil in said biomass comprises at least 70% DHA.
 18. Thebiomass of microorganisms according to claim 11, wherein the microbialoil in said biomass comprises docosapentaenoic acid (DPA) and thecombined DHA and DPA content in said microbial oil is at least 80% inrelation to the total mass of fat.
 19. The biomass of microorganismsaccording to claim 11, wherein the microbial oil in said biomasscomprises docosapentaenoic acid (DPA) and the DHA/DPA ratio in saidmicrobial oil is at least 4.